Rubella (German measles) is usually a benign childhood infection, but rubella virus (RV) can cause a persistent infection of the brain called progressive rubella panencephalitis (ref. 40,51--the literature references are listed at the end of the specification). RV has been isolated from synovial cells of some patients with juvenile rheumatoid arthritis (ref. 8,13). Several live attenuated rubella vaccines have been introduced since 1969 (ref. 2,41). Immunization of infants and susceptible women of child-bearing age against rubella virus is now a standard public health measure. However, there are serious medical concerns with the use of live attenuated rubella virus vaccine for routine immunization. These concerns include the risk of congenital infection of the fetus resulting in diabetis-related diseases (ref. 44) and rubella-associated arthritis following rubella vaccination (ref. 8,47), as well as the possibility of re-infection of vaccinees by wild-type RV due to antigenic differences between wild-type and vaccine virus strains (ref. 11,21). In addition to these problems, rubella virus grows to a relatively low titer in tissue cultures and its structural proteins are difficult to purify (ref. 27). Therefore, there is a clear requirement for preparing a non-infectious rubella vaccine by alternative means, such as recombinant DNA technology and peptides synthesis. Research efforts have recently focused on characterizing both the viral genome and the host immune responses.
RV is the sole member of the genus Rubivirus in the Togavirus family (ref. 29). The primary sequences of the rubella virus structural proteins decoded from cDNA clones have been reported (ref. 10). The RV virion contains an RNA genome enclosed within an icosahedral capsid composed of multiple copies of a basic capsid protein C of 33 kDa (ref. 38). Surrounding this nucleocapsid is a lipid bilayer in which viral glycoproteins E1 (58 kDa) and E2 (42 to 47 kDa) are embedded (ref. 38,43). Glycoprotein E1 has been shown to contain hemagglutinin and virus neutralization epitopes (ref. 50). The data accumulated to date suggest that none of these E1 neutralization epitopes is appropriate for use in a vaccine against RV since they failed to elicit high-titer neutralizing antibody responses against RV in animal studies. E2-specific antibodies are capable of neutralizing viral infection in vitro (ref. 17). However, neutralization epitopes of the E2 protein have not yet been mapped.
Studies have been carried out to characterize the specificity of the antibody response against rubella virus. The RV-specific IgM response is widely used for the diagnosis of recent rubella virus infection (ref. 19,37), and the production of RV-specific IgA antibodies has been shown to be important in the prevention of reinfection (ref. 19). Most of the RV-specific IgM antibodies react with the E1 protein while most of the IgA antibodies react with the C protein (ref. 42). IgG antibody responses can be elicited by all the structural proteins (ref. 30,42).
There is little known about the cellular immune response to RV structural proteins, although both T-helper cell proliferation (ref. 4, 22 to 24, 28, 49) and cytolytic T lymphocyte (CTL) responses (ref. 49) can be detected during viral infection. Studies cited above have neither identified the T-helper determinants nor the CTL epitopes of the rubella structural proteins. Therefore, the identification of these T-cell epitopes (T-helper and CTL) may lead to the design of a safe and effective rubella vaccine.
Methods for inducing immunity against disease are constantly improving and the current trend is to use smaller and well-defined materials as antigens. The objective is to minimize or eliminate the potential side-effects caused by certain native immunogens, while preserving both their immunogenicity and ability to confer protection against disease. Recent studies have indicated that immunization of experimental animals with synthetic peptides representing specific regions of viral or bacterial proteins can induce immune responses specific against the parent proteins, and neutralize their biological functions (ref. 3,18,25,33 to 36). Thus, synthetic peptides are potential candidate antigens for the production of inexpensive and safe vaccines against infectious diseases. Recent progress in fundamental immunology has revealed that, to be efficacious, immunogens should contain two distinct functional domains. One domain is responsible for B-cell recognition and antibody production and the second domain induce T-helper cell activity. Certainly, rubella-specific cytotoxic lymphocyte (CTL) epitopes should be included in the final synthetic vaccine constructs to provide necessary cellular immunity to rubella disease. A recent study (ref. 1) has demonstrated that peptides could prime mice for a CTL responses in vivo. Hence, a safe and effective synthetic peptide vaccine is conceivable.
To design a synthetic peptide-based rubella vaccine, the RV-specific CTL determinants, the viral neutralization B-cell epitopes (BE) and the functional T-helper epitopes of individual viral proteins must be identified. For a synthetic construct to be potent and efficacious, both functional T-helper and B-cell epitopes should be present. To this end, different T-B tandem synthetic peptides, both hybrid and chimeric, are synthesized to determine whether a preferential spatial relationship between T-helper determinants (THD) and B-cell epitopes in a synthetic construct is required for immunogenicity. In addition, the formulation of these synthetic constructs either with adjuvants or lipopeptides are studied to enhance immune responses.
The presentation of an appropriate processed T-cell epitope in the appropriate MHC context and the availability of an appropriate T-cell repertoire are necessary for induction of a cellular immune response. These factors vary among individuals of an outbred population and differences in T-cell responses to subunit vaccines have been reported (Zevering et al. Immunology, 2:945-955, 1990). Other host factors, such as a possible selective T-cell tolerance to RV, also might influence antigen recognition by T-cells. Therefore, the identification of epitopes recognized by the T-cells of individuals of different genetic background and diverse immunologic experience with RV infection or immunization is important for the design of an effective synthetic vaccine.
To map the functional epitopes of rubella viral proteins, we have synthesized 28, 15 and 11 overlapping synthetic peptides covering most of the E1, E2 and C protein sequences, respectively (Tables 1, 2 and 3 below). The length of synthetic peptides was selected on the basis of their high index of hydrophilic .beta.-turns as judged by secondary structure prediction analysis according to the conventional algorithms (ref. 9,12,20) (FIGS. 1 to 3). Such segments are likely to be surface-exposed and antigenic. Long peptides were synthesized to better mimic the native epitopes of the protein as suggested by the work of Van Regenmortel (ref. 48). An additional cysteine residue was added to either the N-terminal or the C-terminal end of the peptides for conjugation purposes.